Abstract
Three positive selection procedures were developed for the isolation of plasmid-encoded mutants which were defective in the mannitol enzyme II (IIMtl) of the phosphotransferase system (mtlA mutants). The mutants were characterized with respect to the following properties: (i) fermentation, (ii) transport, (iii) phosphoenolpyruvate(PEP)-dependent phosphorylation, and (iv) mannitol-1-phosphate-dependent transphosphorylation of mannitol. Cell lysis in response to indole acrylic acid, which causes the lethal overexpression of the plasmid-encoded mtlA gene, was also scored. No correlation was noted between residual IIMtl activity in the mutants and sensitivity to the toxic effect of indole acrylic acid. Plasmid-encoded mutants were isolated with (i) total or partial loss of all activities assayed, (ii) nearly normal rates of transphosphorylation but reduced rates of PEP-dependent phosphorylation, (iii) nearly normal rates of PEP-dependent phosphorylation but reduced rates of transphosphorylation, and (iv) total loss of transport activity but substantial retention of both phosphorylation activities in vitro. A mutant of this fourth class was extensively characterized. The mutant IIMtl was shown to be more thermolabile than the wild-type enzyme, it exhibited altered kinetic behavior, and it was shown to arise by a single nucleotide substitution (G-895----A) in the mtlA gene, causing a single amino acyl substitution (Gly-253----Glu) in the permease. The results show that a single amino acyl substitution can abolish transport function without abolishing phosphorylation activity. This work serves to identify a site which is crucial to the transport function of the enzyme.
Full text
PDF![1290](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cd1d/210905/29447eef83bf/jbacter00181-0274.png)
![1291](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cd1d/210905/48290da44f0a/jbacter00181-0275.png)
![1292](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cd1d/210905/3c0cd074af09/jbacter00181-0276.png)
![1293](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cd1d/210905/9d1901db1c63/jbacter00181-0277.png)
![1294](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cd1d/210905/7a1c21f2f959/jbacter00181-0278.png)
![1295](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cd1d/210905/2c62c587e6cf/jbacter00181-0279.png)
![1296](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cd1d/210905/1040517cc0ea/jbacter00181-0280.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bramley H. F., Kornberg H. L. Nucleotide sequence of bglC, the gene specifying enzymeIIbgl of the PEP:sugar phosphotransferase system in Escherichia coli K12, and overexpression of the gene product. J Gen Microbiol. 1987 Mar;133(3):563–573. doi: 10.1099/00221287-133-3-563. [DOI] [PubMed] [Google Scholar]
- Bramley H. F., Kornberg H. L. Sequence homologies between proteins of bacterial phosphoenolpyruvate-dependent sugar phosphotransferase systems: identification of possible phosphate-carrying histidine residues. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4777–4780. doi: 10.1073/pnas.84.14.4777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jacobson G. R., Kelly D. M., Finlay D. R. The intramembrane topography of the mannitol-specific enzyme II of the Escherichia coli phosphotransferase system. J Biol Chem. 1983 Mar 10;258(5):2955–2959. [PubMed] [Google Scholar]
- Jacobson G. R., Lee C. A., Leonard J. E., Saier M. H., Jr Mannitol-specific enzyme II of the bacterial phosphotransferase system. I. Properties of the purified permease. J Biol Chem. 1983 Sep 10;258(17):10748–10756. [PubMed] [Google Scholar]
- Jacobson G. R., Lee C. A., Saier M. H., Jr Purification of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system. J Biol Chem. 1979 Jan 25;254(2):249–252. [PubMed] [Google Scholar]
- Jacobson G. R., Tanney L. E., Kelly D. M., Palman K. B., Corn S. B. Substrate and phospholipid specificity of the purified mannitol permease of Escherichia coli. J Cell Biochem. 1983;23(1-4):231–240. doi: 10.1002/jcb.240230120. [DOI] [PubMed] [Google Scholar]
- Kundig W., Roseman S. Sugar transport. I. Isolation of a phosphotransferase system from Escherichia coli. J Biol Chem. 1971 Mar 10;246(5):1393–1406. [PubMed] [Google Scholar]
- Lee C. A., Jacobson G. R., Saier M. H., Jr Plasmid-directed synthesis of enzymes required for D-mannitol transport and utilization in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7336–7340. doi: 10.1073/pnas.78.12.7336. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee C. A., Saier M. H., Jr Mannitol-specific enzyme II of the bacterial phosphotransferase system. III. The nucleotide sequence of the permease gene. J Biol Chem. 1983 Sep 10;258(17):10761–10767. [PubMed] [Google Scholar]
- Lee C. A., Saier M. H., Jr Use of cloned mtl genes of Escherichia coli to introduce mtl deletion mutations into the chromosome. J Bacteriol. 1983 Feb;153(2):685–692. doi: 10.1128/jb.153.2.685-692.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leonard J. E., Saier M. H., Jr Genetic dissection of catalytic activities of the Salmonella typhimurium mannitol enzyme II. J Bacteriol. 1981 Feb;145(2):1106–1109. doi: 10.1128/jb.145.2.1106-1109.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leonard J. E., Saier M. H., Jr Mannitol-specific enzyme II of the bacterial phosphotransferase system. II. Reconstitution of vectorial transphosphorylation in phospholipid vesicles. J Biol Chem. 1983 Sep 10;258(17):10757–10760. [PubMed] [Google Scholar]
- Novotny M. J., Reizer J., Esch F., Saier M. H., Jr Purification and properties of D-mannitol-1-phosphate dehydrogenase and D-glucitol-6-phosphate dehydrogenase from Escherichia coli. J Bacteriol. 1984 Sep;159(3):986–990. doi: 10.1128/jb.159.3.986-990.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Postma P. W., Lengeler J. W. Phosphoenolpyruvate:carbohydrate phosphotransferase system of bacteria. Microbiol Rev. 1985 Sep;49(3):232–269. doi: 10.1128/mr.49.3.232-269.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saier M. H., Jr, Feucht B. U., Mora W. K. Sugar phosphate: sugar transphosphorylation and exchange group translocation catalyzed by the enzyme 11 complexes of the bacterial phosphoenolpyruvate: sugar phosphotransferase system. J Biol Chem. 1977 Dec 25;252(24):8899–8907. [PubMed] [Google Scholar]
- Saier M. H., Jr, Grenier F. C., Lee C. A., Waygood E. B. Evidence for the evolutionary relatedness of the proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase system. J Cell Biochem. 1985;27(1):43–56. doi: 10.1002/jcb.240270106. [DOI] [PubMed] [Google Scholar]
- Schnetz K., Toloczyki C., Rak B. Beta-glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes. J Bacteriol. 1987 Jun;169(6):2579–2590. doi: 10.1128/jb.169.6.2579-2590.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamada M., Saier M. H., Jr Glucitol-specific enzymes of the phosphotransferase system in Escherichia coli. Nucleotide sequence of the gut operon. J Biol Chem. 1987 Apr 25;262(12):5455–5463. [PubMed] [Google Scholar]
- Yamada M., Saier M. H., Jr Physical and genetic characterization of the glucitol operon in Escherichia coli. J Bacteriol. 1987 Jul;169(7):2990–2994. doi: 10.1128/jb.169.7.2990-2994.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]